Sebastien de Feraudy

A minority of foci or pan-nuclear apoptotic staining of γH2AX in the S phase after UV damage contain DNA double-strand breaks

UV irradiation induces histone variant H2AX phosphorylated on serine 139 (γH2AX) foci and high levels of pan-nuclear γH2AX staining without foci, but the significance of this finding is still uncertain. We examined the formation of γH2AX and 53BP1 that coincide at sites of double-strand breaks (DSBs) after ionizing radiation. We compared UV irradiation and treatment with etoposide, an agent that causes DSBs during DNA replication. We found that during DNA replication, UV irradiation induced at least three classes of γH2AX response: a minority of γH2AX foci colocalizing with 53BP1 foci that represent DSBs at replication sites, a majority of γH2AX foci that did not colocalize with 53BP1 foci, and cells with high levels of pan-nuclear γH2AX without foci of either γH2AX or 53BP1. Ataxia-telangiectasia mutated kinase and JNK mediated the UV-induced pan-nuclear γH2Ax, which preceded and paralleled UV-induced S phase apoptosis. These high levels of pan-nuclear γH2AX were further increased by loss of the bypass polymerase Pol η and inhibition of ataxia-telangiectasia and Rad3-related, but the levels required the presence of the damage-binding proteins of excision repair xeroderma pigmentosum complementation group A and C proteins. DSBs, therefore, represent a small variable fraction of UV-induced γH2AX foci dependent on repair capacity, and they are not detected within high levels of pan-nuclear γH2AX, a preapoptotic signal associated with ATM- and JNK-dependent apoptosis during replication. The formation of γH2AX foci after treatment with DNA-damaging agents cannot, therefore, be used as a direct measure of DSBs without independent corroborating evidence.

Evaluation of CD10 and procollagen 1 expression in atypical fibroxanthoma and dermatofibroma.

Atypical fibroxanthoma (AFX) (dermal pleomorphic sarcoma) remains a somewhat controversial entity. Some authors have averred that AFX is a fiction, suggesting that such lesions merely represent misclassified examples of spindled squamous cell carcinoma. In addition, the immunoperoxidase confirmation of AFX has been less than straightforward and has historically been approached as a diagnosis of exclusion because of the lack of sensitivity and specificity of available “positive” reagents. Procollagen 1 (PC1) and CD10 represent recently developed immunoperoxidase reagents that have been forwarded as useful in this setting, and we sought to characterize our experience, both to confirm the utility of these antibodies and to compare them. Our investigation included 3 separate data sets. Group 1 consisted of a retrospective review of 98 consecutive cases in which PC1 was used in the evaluation of dermatopathology specimens in routine practice during a 13-month interval. Group 2 consisted of a direct comparison of 11 AFX, 11 dermatofibroma (DF), and 7 epithelioid dermatofibroma (EDF) using the CD10 reagent on cases identified by database search. Group 3 consisted of a retrospective review of 47 cases in which CD10 was used in routine practice during a 10-month interval. Group 1 included 47 AFX, 13 carcinomas, and 6 melanomas. PC1 expression was observed in 45 of 47 AFX (96%), with a strong reaction in 78% of cases. Among a comparison group of carcinomas, 13 of 13 displayed strong keratin immunopositivity and 11 of 13 (85%) lacked PC1 expression whereas 2 showed focal weak labeling. Six of six melanomas exhibited avid S100 expression and none labeled with PC1. In group 2, strong CD10 immunoreactivity was present in 11 of 11 AFX. Similarly, 11 of 11 DFs were also positive. In contrast, 6 of 7 cases of EDF lacked CD10 expression. Group 3 included 38 AFX and 9 miscellaneous spindle cell proliferations. Of the 38 AFX, 37 (97%) labeled with CD10 and in 34 (92%) the reaction was strong. PC1 immunostaining was also completed in 34 of 38 AFX from group 3 and 27 (79%) cases showed positive labeling. Our results confirm that both PC1 and CD10 can be used as positive markers of AFX. We believe that CD10 and PC1 immunostaining can be used as a useful adjunct to supplement the diagnosis of AFX, within the context of an immunoperoxidase panel. Not surprisingly, CD10 expression is also common in DF, a benign analog of AFX, with the exception of its epithelioid variant. In direct head-to-head comparison, our experience indicates that the staining of AFX with CD10 is more avid than that observed with PC1. Lastly, out data includes over 80 examples of AFX, <5% of which showed keratin labeling. Given a general lack of keratin expression, it seems unlikely that AFX merely represents poorly differentiated squamous carcinoma.

Intradermal nodular fasciitis: a rare lesion analyzed in a series of 24 cases.

Nodular fasciitis, a benign myofibroblastic proliferation that usually occurs in the subcutaneous tissues of the upper extremities, trunk, as well as head and neck of young adults, is not widely recognized to arise primarily within the dermis. Here, we examined the clinicopathologic and immunohistochemical features of a series of 24 cases of intradermal nodular fasciitis retrieved from consult files. Clinical follow-up was obtained from medical records and referring physicians. Fourteen patients were females and 10 were males, with age ranging from 8 to 77 years (mean 27.5u2009y and median 22.5u2009y). Most patients presented with a rapidly growing painless solitary mass. Grossly, the lesions were solid, nodular, rubbery, or firm (mean size 1.96u2009cm and median 1.3u2009cm). Nine cases (37.5%) arose on limbs, 9 cases (37.5%) on the trunk, and 6 (25%) on the head and neck. All tumors were situated primarily in the dermis and were well circumscribed but unencapsulated. One case showed ill-defined margins. The epidermis was ulcerated in 11 cases. All tumors were composed of cytologically bland, uniform plump spindle cells with elongated, tapering nuclei, vesicular chromatin, small nucleoli, and palely eosinophilic cytoplasm, with no significant cytologic atypia or pleomorphism. These cells were arranged in short-intersecting bundles within a focally microcystic myxoid stroma, containing extravasated red blood cells and scattered lymphocytes. Mitoses ranged from 1 to 6 per 10 high-power fields (mean 2.5). All cases examined were diffusely positive for smooth muscle actin. Only one tumor “recurred” locally. No lesion metastasized. In summary, intradermal nodular fasciitis occurs most commonly on the limbs and trunk of young adults, shows morphologic features similar to nodular fasciitis at conventional sites and should not be confused with sarcoma.

Diagnosing Xeroderma Pigmentosum Group C by Immunohistochemistry

Xeroderma pigmentosum (XP) is a group of rare inherited human neurocutaneous diseases, and the group C (XPC) is the major group of patients with XP in Europe, North America, and South America. Current molecular diagnostic methods for XP require specialized, expensive, and time-consuming UV sensitivity and DNA repair assays followed by gene sequencing. To determine whether immunohistochemistry (IHC) would be a robust alternative method to diagnose patients with XPC, we stained sections of paraffin-embedded skin biopsies for XPC by IHC, using 69 archived blocks from confirmed or clinically suspect patients with XPA, XPC, XPD, XPE, and without XP. We found that XPC expression was strong in all skin biopsies from patients without (14 of 14) and other patients with XP (4 of 4), whereas XPC expression was lost in all biopsies from confirmed XPC patients (29 of 29). Patches of strong XPC signal could be detected in sun-damaged skin, squamous and basal cell carcinomas from patients with XPC that colocalized with strong expression of p53 and Ki-67. Patients with XPC can therefore be diagnosed by IHC from paraffin-embedded skin biopsies from regions of skin that are without sun damage or sun-induced tumors. IHC is therefore a robust alternative method to diagnose patients with XPC. This fast and inexpensive method should increase the options for the diagnosis of patients with XPC from paraffin-embedded skin biopsies and could be developed for other complementation groups.

Antitumor Activity of miR-1280 in Melanoma by Regulation of Src

MicroRNAs (miRNAs) play a key role in cancer progression by coordinately repressing target genes involved in cell proliferation, migration, and invasion. miRNAs regulate gene expression by repressing translation or directing sequence-specific degradation of complementary mRNA. Here, we report that expression of miR-1280 is significantly suppressed in human melanoma specimens when compared with nevi, and in human melanoma cell lines when compared with cultured normal human melanocytes. The proto-oncogene Src was identified as a target of miR-1280 action. Levels of Src expression were significantly higher in melanoma samples and cell lines than in nevi and normal melanocytes. miR-1280 overexpression significantly suppressed the luciferase activity of reporter plasmids containing the full-length 3 untranslated region of Src. miR-1280-mediated suppression of Src led to substantial decreases in melanoma cell proliferation, cell cycle progression, invasion, as well as induced melanoma cell apoptosis. The effects of miR-1280 overexpression on melanoma cell proliferation and growth were reversed by Src overexpression. Intratumoral delivery of miR-1280 significantly suppressed melanoma cell growth in vivo. Our results demonstrate a novel role for miR-1280 as a tumor suppressor in melanoma, identify the Src signaling pathway as a target of miR-1280 action, and suggest a potential therapeutic role for miR-1280 in melanoma.

Localized pretibial bullous pemphigoid arising in a patient on pembrolizumab for metastatic melanoma

Author(s): Amber, Kyle T; Valdebran, Manuel; Lu, Yuxin; De Feraudy, Sebastien; Linden, Kenneth G

ATR Mutations Promote the Growth of Melanoma Tumors by Modulating the Immune Microenvironment

Melanomas accumulate a high burden of mutations that could potentially generate neoantigens, yet somehow suppress the immune response to facilitate continued growth. In this study, we identify a subset of human melanomas that have loss-of-function mutations in ATR, a kinase that recognizes and repairs UV-induced DNA damage and is required for cellular proliferation. ATR mutant tumors exhibit both the accumulation of multiple mutations and the altered expression of inflammatory genes, resulting in decreased Txa0cell recruitment and increased recruitment of macrophages known to spur tumor invasion. Taken together, these studies identify a mechanism by which melanoma cells modulate the immune microenvironment to promote continued growth.

Vascularization and innervation of slits within polydimethylsiloxane sheets in the subcutaneous space of athymic nude mice

Success of cell therapy in avascular sites will depend on providing sufficient blood supply to transplanted tissues. A popular strategy of providing blood supply is to embed cells within a functionalized hydrogel implanted within the host to stimulate neovascularization. However, hydrogel systems are not always amenable for removal post-transplantation; thus, it may be advantageous to implant a device that contains cells while also providing access to the circulation so retrieval is possible. Here we investigate one instance of providing access to a vessel network, a thin sheet with through-cut slits, and determine if it can be vascularized from autologous materials. We compared the effect of slit width on vascularization of a thin sheet following subcutaneous implantation into an animal model. Polydimethylsiloxane sheets with varying slit widths (approximately 150, 300, 500, or 1500u2009µm) were fabricated from three-dimensional printed molds. Subcutaneous implantation of sheets in immunodeficient mice revealed that smaller slit widths have evidence of angiogenesis and new tissue growth, while larger slit widths contain native mature tissue squeezing into the space. Our results show that engineered slit sheets may provide a simple approach to cell transplantation by providing a prevascularized and innervated environment.

The RhoJ-BAD signaling network: An Achilles' heel for BRAF mutant melanomas.

Genes and pathways that allow cells to cope with oncogene-induced stress represent selective cancer therapeutic targets that remain largely undiscovered. In this study, we identify a RhoJ signaling pathway that is a selective therapeutic target for BRAF mutant cells. RhoJ deletion in BRAF mutant melanocytes modulates the expression of the pro-apoptotic protein BAD as well as genes involved in cellular metabolism, impairing nevus formation, cellular transformation, and metastasis. Short-term treatment of nascent melanoma tumors with PAK inhibitors that block RhoJ signaling halts the growth of BRAF mutant melanoma tumors in vivo and induces apoptosis in melanoma cells in vitro via a BAD-dependent mechanism. As up to 50% of BRAF mutant human melanomas express high levels of RhoJ, these studies nominate the RhoJ-BAD signaling network as a therapeutic vulnerability for fledgling BRAF mutant human tumors.

CD30 Positive Lymphomatoid Angiocentric Drug Reactions: Characterization of a Series of 20 Cases.

Introduction: Lymphomatoid drug reactions are atypical T cell cutaneous lymphocytic infiltrates induced by pharmacological therapy. Due to phenotypic abnormalities, clonality, and their close clinical and morphologic resemblance to T cell lymphomas, these eruptions have been categorized as drug-associated reversible granulomatous T cell dyscrasias. Design: A total of 20 cases were encountered in which a diagnosis of CD30+ lymphomatoid drug reaction was rendered. Results: There were 11 women and 9 men ranging from 31 to 86 years of age presenting with a sudden onset often generalized cutaneous papular eruption. Two patients had vasculitic lesions. In all cases, a positive drug history was elicited and in most the initiation of the drug was temporally associated with the cutaneous eruption. Among the implicated drugs were statins (6 cases), immunomodulators (4 cases), ACE inhibitors (3 cases), antibiotics (3 cases), chemotherapy agents (3 cases), and antidepressants (1 case). Biopsies demonstrated a similar morphology, namely a superficial angiocentric lymphocytic infiltrate containing many immunoblasts. Tissue eosinophilia, interface dermatitis, and supervening eczematous changes in the overlying epidermis were observed in most cases. In all cases, the angiocentric infiltrate was highlighted by CD3, CD30, and CD4. Cytotoxic protein granule expression or monoclonality was not observed. In all cases, there was improvement or complete regression of the eruption upon drug modulation. Conclusion: The CD30 positive lymphomatoid angiocentric drug reaction poses a diagnostic challenge because of its close resemblance to type A lymphomatoid papulosis and potential confusion with a peripheral T cell lymphoma with large cell transformation.